A major impetus for developing this program project is to consolidate and coordinate our efforts to establish a core laboratory designed to facilitate the production and purification of large multi- subunit regulatory complexes involved in gene transcription. Consequently, the establishment of a highly efficient, well integrated and interdependent core facility to meet these considerable experimental challenges will be critical to the success of this Program Project. We have therefore subdivided the Core Lab into 3 distinct divisions, each with its own set of defined specific aims: 1) Construction of expression systems and production of tagged subunit polypeptides; 2) Generation and production of monoclonal antibodies directed against mapped epitopes of target subunits; and 3) Development of small- and large-scale antibody affinity purification for multi-subunit complexes. Indeed, each of the 3 projects proposed in this program will require the large-scale production and purification of molecular machines. These represent the principal protein complexes targeted for functional and structural studies using a combination of cryo-EM/single particle reconstruction, X-ray crystallography, and single molecule FRET analysis. In each of the 3 projects, overlapping sets of proteins and protein complexes such as human TFIID, CRSP, ARC, PBAF, RNA polymerase II, the pre-initiation complex (PIC), and their attendant sub-complexes, as well as activator binding factors, will be the primary subjects of study. In most cases, these different multi- subunit assemblages will be isolated from human cells, but in some instances, depending on the exact nature of the experimental strategy, we will also make use of analogous functional complexes isolated from Drosophila. Regardless of the original cell type or species of the starting material, a common set of experimental strategies and related protocols will need to be developed in order to carry out the structural studies that form the scientific basis of this program project.